The long-term goal of the studies proposed here is to understand the structural basis for normal activation[unreadable] of Notch receptors, to define the biochemical mechanism underlying pathophysiologic activation of Notch by[unreadable] mutations found in T cell acute lymphocytic leukemia/lymphoma (T-ALL), and ultimately to harness this[unreadable] knowledge for the treatment of T-ALL.[unreadable] To understand how the non-covalently associated subunits of Notch proteins are normally held together,[unreadable] find out how cancer-associated mutations increase Notch signaling, and elucidate how the Notch[unreadable] transcriptional activation complex is assembled, we propose the following specific aims:[unreadable] Aim 1. Determine the structure, dynamics, and interactions of a Notch heterodimerization domain.[unreadable] Aim 2. Determine how tumor-derived heterodimerization domain mutations activate Notch.[unreadable] Aim 3. Determine the structural basis underlying activation of transcription by Notch receptors.[unreadable] 3A. Solve the structure of a Notch transcriptional activation complex or a key subcomplex containing the[unreadable] Notch ankyrin repeat domain.[unreadable] 3B. Identify small molecules that prevent assembly of the Notch transcriptional activation complex.[unreadable] The structural studies proposed in this proposal will fill a major gap in our current understanding of Notch[unreadable] signaling. The proposed NMR studies of a Notch heterodimerization domain will reveal the nature of an[unreadable] intramolecular interface that is crucial for maintaining Notch in its resting conformation, and that is now[unreadable] known to harbor activating mutations found frequently in Notch-dependent human T-ALLs. The proposed Xray[unreadable] crystallographic studies of Notch transcriptional activation complexes will uncover how the ankyrin repeat[unreadable] domain of Notch cooperates with the CSL transcription factor to recruit the mastermind co-activator. These[unreadable] advances will identify candidate residues for mutational studies designed to probe the importance of specific[unreadable] ankyrin repeat contacts in coordinating mastermind recruitment, and in binding to CSL.[unreadable] Finally, the availability of purified ternary complexes of Notch, Mastermind-like polypeptides and CSL[unreadable] bound to cognate DNA creates an opportunity for us to identify small molecules that disrupt the complex,[unreadable] with potential future utility in probing Notch signaling in vivo, and as compound leads for targeted therapy in[unreadable] T-ALL. Because of the broad importance of NOTCH in differentiation and proliferation, interventions that[unreadable] prevent NOTCH signaling may not only lead to new forms of treatment for T-ALL, but manipulation of[unreadable] NOTCH activity may also be of general value in management of breast and other cancers as well.